At least three major, noninterconvertible, tetrameric isozymes of pyruvate kinase (EC 2.7.1.40) occur in mammals. The isozymes can be distinguished electrophoretically and have different kinetic properties that presumably allow each to function optimally in the tissue where it is found. Isozymic hybrids can be produced in vitro or occur in vivo when two subunit types are synthesized simultaneously in the same cell. We are examining the chemical, structural, and regulatory properties of the pyruvate kinase isozymes in order to better understand how they function in normal tissue and under pathological conditions. Our approach is to (a) use kinetic and electrophoretic procedures to examine the hormonal, nutritional, and develomental factors that determine isozymic patterns in liver and muscle; (b) determine the rate of enzyme synthesis and degradation via incorporation of radioatctive amino acids into specific immunoprecipitable protein; (c) use fluorescence, circular dichroism, and enzyme kinetics to study the process by which unfolded polypeptide chains form enzymatically active tetramers; (d) kinetically characterize hybrid isozymes formed from subunits having very different kinetic properties; and (e) use chemical modification with or without substrate protection followed by peptide cleavage and sequencing to determine the amino acid residues that are important in substrate binding and catalysis.